Method for detecting DNA methylation of OTX1 gene and PENK gene and application

Abstract

The invention belongs to the field of gene engineering, and provides a primer and probe combination, a kit and a method for detecting DNA methylation of an OTX1 gene and a PENK gene. According to the invention, a high-sensitivity 3 'UTR region of the OTX1 gene and a first exon region of the PENK gene are selected, a methylated and non-methylated primer and probe combination is designed, and the OTX1 gene and the PENK gene are subjected to joint detection, so that the detection sensitivity and accuracy are greatly improved. Meanwhile, the TaqMan probe with two marked ends is selected, so that the sensitivity, specificity and accuracy of detection are improved, and the kit has a very good clinical application value.

Description

Technical Field

[0001] The present invention relates to molecular detection technology, in particular to a method for detecting DNA methylation of OTX1 gene and PENK gene and application thereof, belonging to the field of genetic engineering. Background Art

[0002] DNA methylation is a well-known epigenetic mechanism. Hypomethylation activates gene transcription, while hypermethylation inhibits gene transcription, leading to gene silencing.

[0003] Methylation imbalance is one of the most common epigenetic changes in tumors. Its occurrence is mainly manifested by a decrease in the methylation level of oncogenes in the entire genome and an abnormal increase in the local methylation level of the CpG island of tumor suppressor genes.

[0004] DNA methylation is a good biomarker for cancer screening. Abnormal DNA methylation is an early event in carcinogenesis. It is tissue-specific and chemically as stable as DNA, and can be stably detected in plasma and urine. At the same time, DNA methylation markers are sensitive, specific, and easy to analyze, making them potential routine clinical cancer biomarkers. DNA methylation provides a new way to detect early and recurrent cancers of various types.

[0005] OTX1 (Orthodenticle Homeobox 1) belongs to the OTX transcription factor family. The transcription factor it encodes has high affinity with the TAATCC / T elements on the target gene.

[0006] As an important transcription factor, OTX1 participates in the development of the cerebral cortex, sensory organs, early human fetal retina and mammary gland, and is also involved in the progression of various tumors. OTX1 is overexpressed in various cancers, including mammary blastoma, breast cancer, colorectal cancer and hepatocellular carcinoma. Therefore, OTX1 may be closely related to the development and progression of human cancer. OTX1 promotes the proliferation and migration of tumor cells, promotes the growth and movement of bladder cancer cells in vitro, and accelerates the cell cycle process. An international multicenter prospective study showed that the methylation of OTX1 together with FGFR3 and TERT mutations can predict the recurrence of non-muscle invasive bladder cancer (NMIBC). In addition, OTX1 is a potential prognostic biomarker for bladder cancer (BCa), so it is of great significance to detect the methylation level of OTX1.

[0007] PENK (Proenkephalin) is a proprotein that can produce enkephalin after being cleaved by proteases. Enkephalin plays a vital role in the central nervous system. Enkephalin can bind to the receptors of the central nervous system, regulate the body's life activities through the hypothalamus-pituitary-adrenal signal axis, increase the activity of natural killer cells and macrophages, and improve the body's immune response and anti-tumor capabilities.

[0008] PENK methylation is frequently found in patients with bladder cancer but rarely in patients with non-bladder cancer hematuria, so detecting the methylation level of PENK can be used as a useful diagnostic tool for detecting bladder cancer.

[0009] At present, the main methods for detecting methylation include BSP (sodium bisulfite sequencing), COBRA (sodium bisulfite treatment restriction enzyme digestion), MSP (methylation-specific PCR), DHPLC (distinct high performance liquid chromatography) and Methylight (methylation fluorescence detection). Among them, BSP is relatively expensive and not suitable for large-scale clinical application; COBRA can only obtain methylation at special enzyme cutting sites; MSP is relatively simple, but can only perform qualitative analysis; DHPLC experiments are complex and the instruments are expensive. Compared with the above methods, Methylight is a DNA methylation detection method with high sensitivity, simple operation, low restriction, moderate cost, and suitable for clinical application. However, for Methylight detection, the selection of detection genes and detection primers and probes is crucial. Summary of the invention

[0010] In view of the defects in the prior art, the present invention provides a method for detecting the DNA methylation levels of OTX1 gene and PENK gene by using methylation fluorescence detection (Methylight) technology.

[0011] To this end, on one hand, the present invention provides a primer and probe combination, which comprises the primer and probe combination described in groups 1-5, wherein group 1 consists of primers shown in SEQ ID NO.4-5 and probes shown in SEQ ID NO.6, group 2 consists of primers shown in SEQ ID NO.7-8 and probes shown in SEQ ID NO.9, and group 1 and group 2 are used to detect DNA methylation of the OTX1 gene; group 3 consists of primers shown in SEQ ID NO.10-11 and probes shown in SEQ ID NO.12, group 4 consists of primers shown in SEQ ID NO.13-14 and probes shown in SEQ ID NO.15, and group 3 and group 4 are used to detect DNA methylation of the PENK gene; group 5 consists of primers shown in SEQ ID NO.16-17 and probes shown in SEQ ID NO.18, and is used to detect DNA methylation of the internal reference gene ACTB gene.

[0012] In a preferred embodiment of the present invention, the primer and probe combination of the present invention consists of the primer and probe combination described in Groups 1 to 5,

[0013] Another aspect of the present invention provides use of the primer and probe combination of the present invention in preparing a kit.

[0014] Another aspect of the present invention provides a kit, which contains the primer and probe combination of the present invention. In a preferred embodiment of the present invention, the kit of the present invention also contains a reagent for performing methylation fluorescence detection (Methylight) on a sample.

[0015] Another aspect of the present invention provides a PCR amplification method for performing methylation fluorescence detection (Methylight) on a sample, which is a three-step method, comprising the following steps:

[0016] 1. 95℃, pre-denaturation, 5 minutes,

[0017] 2. 95℃, denaturation, 15 seconds,

[0018] 3. Annealing at 60°C for 1 minute;

[0019] Step 2 and step 3 were repeated 40 times.

[0020] The primer and probe combination of the present invention or the kit of the present invention is used.

[0021] Another aspect of the present invention provides use of the primer and probe combination of the present invention, the kit of the present invention, or the PCR amplification method of the present invention in detecting DNA methylation of OTX1 gene and PENK gene.

[0022] In another aspect, the present invention provides a method for detecting DNA methylation of OTX1 gene and PENK gene, comprising the following steps:

[0023] The primer and probe combination of the present invention, the kit of the present invention, or the PCR amplification method of the present invention is used to detect the DNA methylation levels of the OTX1 gene and the PENK gene.

[0024] In a preferred embodiment of the present invention, the method of the present invention comprises the following steps:

[0025] 1. Synthesize the primer and probe combination described in the present invention, or use the primer and probe combination contained in the kit described in the present invention;

[0026] 2. Extract sample DNA and perform bisulfite modification;

[0027] 3. The PCR amplification method described in the present invention is used to detect the DNA methylation levels of the OTX1 gene and the PENK gene.

[0028] In a preferred embodiment of the present invention, the method of the present invention detects the DNA methylation levels of the OTX1 gene and the PENK gene by methylation fluorescence detection (Methylight) technology.

[0029] In a more preferred embodiment of the present invention, the sample DNA is urine DNA from a normal person or a bladder cancer patient.

[0030] The present invention achieves the following beneficial effects by adopting the above technical solution:

[0031] 1. In terms of gene selection, the present invention refers to the data on bladder cancer in the TCGA database, GEO database, Pancan-meQTL database, and HACER database, and after comparison and screening, finally determines the OTX1 gene and PENK gene with high specificity and high sensitivity. In bladder cancer, the CpG islands of the OTX1 gene and the PENK gene are highly methylated. The present invention provides a strong basis for the early diagnosis and evaluation of bladder cancer by determining appropriate detection genes and detecting their methylation status, and has good clinical application value.

[0032] 2. The OTX1 gene and the PENK gene are a new gene combination for bladder cancer. The present invention selects the highly sensitive 3'UTR region of the OTX1 gene and the first exon region of the PENK gene, and designs a combination of methylated and unmethylated primers and probes for the joint detection of the OTX1 gene and the PENK gene, which greatly increases the sensitivity and accuracy of the detection.

[0033] 3. The method of the present invention is a non-invasive, painless and non-invasive detection method suitable for urine, which increases the compliance of the subjects. In addition, in the method of the present invention, a double-end labeled TaqMan probe is selected to improve the sensitivity, specificity and accuracy of the detection.

[0034] In summary, the method of the present invention can provide rapid, sensitive, pollution-free, highly specific, highly sensitive and highly accurate detection results, providing a strong basis for the early diagnosis and evaluation of bladder cancer, and has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the distribution map of CpG dinucleotide sites in one chain in the CpG island region of the OTX1 gene.

[0036] The underlined positions indicate the distribution positions of CpG.

[0037] Figure 2 This is the distribution map of CpG dinucleotide sites in one chain in the CpG island region of the PENK gene.

[0038] The underlined positions indicate the distribution positions of CpG.

[0039] Figure 3 Box plot of the differential expression of OTX1 and PENK genes in bladder cancer.

[0040] Figure 4 The changes in the threshold cycle (CT) values ​​of OTX1 gene and PENK gene under the three-step and two-step qPCR methods. DETAILED DESCRIPTION

[0041] The present application is further described in detail below through the accompanying drawings and embodiments. Through these descriptions, the characteristics and advantages of the present application will become clearer and more specific.

[0042] Unless otherwise specified, the reagents, consumables and instruments involved in the present invention can be obtained through general commercial channels.

[0043] Example 1: Design of primers and probes for detecting DNA methylation levels of OTX1 and PENK genes

[0044] 1. Screening of CG-rich DNA regions in OTX1 and PENK gene sequences

[0045] The complete sequences of OTX1 gene (NC_000002.11 (63276870..63284966)) and PENK gene (NC_000008.10 (57353516..57359200)) were downloaded from the NCBI database, and the sequences of the promoter and the first exon region were marked. The CpGPlot tool of EMBOSS (https: / / www.ebi.ac.uk / Tools / seqstats / emboss_cpgplot / ) was used to input the complete sequence of the target gene and predict the CpG island in the gene.

[0046] Attached Figure 1 The distribution of CpG dinucleotide sites in one chain of the CpG island region of the OTX1 gene is shown, wherein the underlined positions indicate the distribution positions of CpG.

[0047] Attached Figure 2 The distribution of CpG dinucleotide sites in one chain of the CpG island region of the PENK gene is shown, wherein the underlined positions indicate the distribution positions of CpG.

[0048] Since a gene may have multiple CpG islands, we focused on the CpG islands that overlapped with the promoter and the first exon regions, and matched them with the differentially methylated sites of bladder cancer in the TCGA database, GEO database, Pancan-meQTL database, and HACER database. Finally, we screened out the highly sensitive 3'UTR region of the OTX1 gene and the first exon region of the PENK gene based on the matching overlap.

[0049] The screened 3'UTR region of the OTX1 gene is shown as SEQ ID NO.1, and the first exon region of the PENK gene is shown as SEQ ID NO.2.

[0050] The expression differences of OTX1 gene and PENK gene in bladder cancer tissue and normal bladder tissue were analyzed using TCGA database and UALCAN database. Figure 3 shown.

[0051] In the attached Figure 3 There were significant differences in the expression of OTX1 gene and PENK gene in bladder cancer (P<0.01).

[0052] 2. Design of primer and probe combination for detecting DNA methylation of OTX1 gene

[0053] According to the sequence shown in SEQ ID NO.1, the software MethPrimer was used to convert the methylated sequence, and the converted sequence was used to assist in the design of methylated primers, and then Primer5 and Oligo7 were used to assist in the design of probes.

[0054] The OTX1 gene sequence was searched from the NCBI database, and SEQ ID NO.1 was designed as the nucleic acid region for detecting the methylation level of the OTX1 gene in the present invention. A pair of methylation primers and a pair of non-methylation primers were designed based on this region. The probe for detecting the target region was set to be a Taqman probe. The reporter group at the 5' end of the probe for detecting the target region was FAM, and the quencher group at the 3' end was BHQ1. The sequences of the primers and probes are as follows:

[0055] (1) Primer and probe combination for OTX1 gene methylation

[0056] Upstream primer sequence for OTX1-1 methylation-specific PCR:

[0057] 5'-TGTTTTCGATTTTGTTTTACGTTTGTC-3' (SEQ ID NO. 4);

[0058] Downstream primer sequences for OTX1-1 methylation-specific PCR:

[0059] 5'-CGAAACGAAATACGACTCTATACGC-3' (SEQ ID NO.5);

[0060] Probe sequence for OTX1-1 methylation-specific PCR:

[0061] 5'-FAM-ACGAACCTAACCCTAACGCGCCCCAAC-BHQ1-3' (SEQ ID NO. 6).

[0062] (2) Primer and probe combination for OTX1 gene unmethylation

[0063] Upstream primer sequence for OTX1-2 non-methylation specific PCR:

[0064] 5'-GTTTTTGATTTTGTTTTATGTTTGTTG-3' (SEQ ID NO. 7);

[0065] Downstream primer sequences for OTX1-2 unmethylation-specific PCR:

[0066] 5'-AAACAAAATACAACTCTATACACAA-3' (SEQ ID NO. 8);

[0067] Probe sequence for OTX1-2 unmethylation-specific PCR:

[0068] 5'-FAM-ACAAACCTAACCCTAACACACCCCAAC-BHQ1-3' (SEQ ID NO. 9).

[0069] 3. Design of primer and probe combination for detecting DNA methylation of PENK gene

[0070] According to the sequence shown in SEQ ID NO.2, the software MethPrimer was used to convert the methylated sequence, and the converted sequence was used to assist in the design of methylated primers, and then Primer5 and Oligo7 were used to assist in the design of probes.

[0071] The PENK gene sequence was searched from the NCBI database, and SEQ ID NO.2 was designed as the nucleic acid region for detecting the methylation level of the PENK gene in the present invention. A pair of methylation primers and a pair of non-methylation primers were designed based on this region. The probe for the target region was set to be a Taqman probe. The reporter group at the 5' end of the probe for the target region was CY5, and the quencher group at the 3' end was BHQ3. The sequences of the primers and probes are as follows:

[0072] (1) Primer and probe combination for PENK gene methylation

[0073] Upstream primer sequence for PENK methylation-specific PCR:

[0074] 5'-GGTTGTTGTTGTTCGGTTTC-3' (SEQ ID NO. 10);

[0075] Downstream primer sequences for PENK methylation-specific PCR:

[0076] 5'-CGACCGAACGCACTAAAC-3' (SEQ ID NO. 11);

[0077] Probe sequence for PENK methylation-specific PCR:

[0078] 5'-CY5-AACTACACGTCGCGCAATCCTAACTACAT-BHQ3-3' (SEQ ID NO. 12).

[0079] (2) Primer and probe combination for PENK gene unmethylation

[0080] Upstream primer sequence for PENK non-methylation-specific PCR:

[0081] 5'-TGTATTTGGGTTGTTGTTGTTTGGTT-3' (SEQ ID NO. 13);

[0082] Downstream primer sequences for PENK non-methylation-specific PCR:

[0083] 5'-ACCAAACACACTAAACAATAACTACAC-3' (SEQ ID NO. 14);

[0084] Probe sequence for PENK unmethylation-specific PCR:

[0085] 5'-CY5-CACACAATCCTAACTACATTCAACCCACAC-BHQ3-3' (SEQ ID NO. 15).

[0086] 4. Design of primer and probe combination for detecting DNA methylation of the internal reference gene ACTB

[0087] The sequence of the internal reference gene ACTB gene was searched from the NCBI database. SEQ ID NO.3 is the nucleic acid region of the ACTB gene detected by the present invention. The primer and probe set were designed based on this region. The probe for detecting the target region was set to be a Taqman probe. The reporter group at the 5' end of the probe for detecting the target region was VIC, and the quencher group at the 3' end was BHQ1. The sequences of the primer probes were as follows:

[0088] Upstream primer sequence for ACTB methylation-specific PCR:

[0089] 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' (SEQ ID NO. 16);

[0090] Downstream primer sequences for ACTB methylation-specific PCR:

[0091] 5'-AACCAATAAAAACCTACTCCTCCCTTAA-3' (SEQ ID NO. 17);

[0092] Probe sequence for ACTB methylation-specific PCR:

[0093] 5'-VIC-ACCACCACCCAACACACAATAACAAACACA-BHQ1-3' (SEQ ID NO. 18).

[0094] The sequence of the internal reference gene ACTB is a conservative sequence that is not affected by methylation. It is used as a data evaluation for detection to eliminate the interference of experimental operations on the results.

[0095] The sequences of the OTX1-1 gene, PENK-1 gene, and ACTB gene are shown in Table 1 .

[0096] Table 1 Sequences of OTX1-1, PENK-1 and ACTB genes

[0097]

[0098]

[0099] The primer and probe sequences of OTX1-1 gene, PENK-1 gene and ACTB gene are shown in Table 2 .

[0100] Table 2 Primer and probe sequences of OTX1-1, PENK-1 and ACTB genes

[0101]

[0102] Example 2: Extraction and bisulfite modification of urine DNA

[0103] 1. Preparation of urine samples

[0104] (1) Take 40.0-50.0 mL of raw urine, centrifuge at 5000 rpm for 10 min at room temperature, and discard the supernatant until about 1 mL of liquid remains. Transfer the above sample to a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 min at room temperature, and discard the supernatant.

[0105] (2) Add 500 μl of PBS solution to the centrifuge tube, invert and mix three times, centrifuge at room temperature and 10,000 rpm for 2 min, and discard the supernatant.

[0106] (3) Repeat step (2).

[0107] (4) Add 25 μl of PBS solution and resuspend.

[0108] 2. Extraction of DNA from urine sediment

[0109] The genomic DNA from urine sediment was extracted using QIAamp DNA Mini Kit (Cat. No. 51304). For specific procedures, please refer to the kit instructions.

[0110] (1) Add 180 μl of Buffer ATL to the resuspended urine sediment.

[0111] (2) Add 20 μl of proteinase K, vortex to mix, and incubate at 56°C until the tissue is completely lysed. Vortex intermittently during the incubation process or place the centrifuge tube in a shaking water bath to ensure that the sample is fully exposed to Buffer ATL.

[0112] (3) Briefly centrifuge the 1.5 mL centrifuge tube to remove droplets hanging on the inner wall.

[0113] (4) Add 200 μl of Buffer AL, shake for 15 seconds, and place in a 70°C water bath for 10 minutes. Centrifuge briefly to remove the liquid on the inner edge of the centrifuge tube cap.

[0114] (5) Add 200 μl of ethanol (96-100%) to the sample, vortex briefly for 15 seconds to mix, and centrifuge quickly to remove droplets hanging on the inner wall.

[0115] (6) Carefully add the mixture obtained above (including the precipitate) to the QIAamp Mini spin column (without wetting the rim), and place the spin column in a 2 ml collection tube. Close the centrifuge tube cap tightly and centrifuge at 6000×g (8000rpm) for 1 min. Remove the centrifuge tube and place it in a clean 2 ml collection tube (provided in the kit). Discard the collection tube and the liquid in it.

[0116] (7) Carefully add 500 μl of Buffer AW1 to the QIAamp Mini spin column (without wetting the rim). Close the centrifuge tube cap and centrifuge at 6000×g (8000 rpm) for 1 min. Remove the centrifuge tube and place it in a clean 2 ml collection tube (provided in the kit). Discard the collection tube and the liquid in it.

[0117] (8) Carefully add 500 μl of Buffer AW2 to the QIAamp Mini spin column (without wetting the rim). Close the centrifuge tube cap and centrifuge at 20,000 × g (14,000 rpm) for 3 min. Remove the centrifuge tube and place it in a clean 2 ml collection tube (provided in the kit). Discard the collection tube and the liquid in it.

[0118] (9) Place the QIAamp Mini spin column into a new 1.5 ml centrifuge tube (prepare your own), discard the collection tube and the liquid in it, and centrifuge at full speed for 1 min.

[0119] (10) Place the QIAamp Mini spin column in a new 1.5 ml centrifuge tube (self-prepared) and discard the used collection tube. Carefully open the lid, add 200 μl of Buffer AE or sterile water, incubate at room temperature (15-25°C) for 1 min, and then centrifuge at 6000 × g (8000 rpm) for 1 min.

[0120] (11) Repeat the previous step.

[0121] (12) The extracted genomic DNA was tested for DNA concentration using Qubit 2.0 and DNA purity using a UV spectrophotometer (ThermoNanoDrop 2000). 260 / OD 280 The ratio was not less than 1.8. The DNA concentration and DNA purity of each sample are shown in Table 3.

[0122] Table 3 DNA concentration and DNA purity of samples

[0123] sample Qubit (concentration) Thermo 2000(purity) sample-1 22.2ng / ul 1.95 sample-2 16.92ng / ul 1.92 sample-3 26.42ng / ul 1.9 sample-4 32.2ng / ul 1.9 sample-5 17.82ng / ul 1.83 sample-6 23.3ng / ul 2

[0124] 3. Bisulfite modification of the extracted genomic DNA

[0125] Using EZ DNA Methylation-Gold TM Kit (Cat. No.: D5005) for bisulfite modification. For specific operations, refer to the kit instructions.

[0126] (1) Add 130 μl of CT Conversion Reagent to a PCR tube containing 20 μl of DNA sample. Mix the sample by flicking the tube or using a pipette. Vortex and centrifuge to remove the liquid inside the tube cap.

[0127] (2) Place the sample tube on the PCR instrument and follow the steps below:

[0128] 98°C for 10 min, 64°C for 2.5 hrs, store at 4°C (up to 20 hrs).

[0129] (3) Add 600 μl of M-Binding Buffer to the Zymo-Spin IC Column and place the column in the Collection Tube provided in the kit.

[0130] (4) Add the sample (from step 2) to the Zymo-Spin IC Column containing M-Binding Buffer, close the lid, and mix the sample by inversion.

[0131] (5) Centrifuge at full speed (>10,000 × g) for 30 seconds and remove the flow-through.

[0132] (6) Add 200 μl of M-Wash Buffer to the column and centrifuge at full speed for 30 seconds.

[0133] (7) Add 200 μl of M-Desulphonation Buffer to the column and incubate at room temperature (20°C-30°C) for 15-20 minutes. After incubation, centrifuge at full speed for 30 seconds.

[0134] (8) Add 200 μl of M-Wash Buffer to the column, centrifuge at full speed for 30 seconds, add another 200 μl of M-Wash Buffer, and centrifuge for 30 seconds.

[0135] (9) Place the column in a 1.5 ml microcentrifuge tube, add 10 μl of M-Elution Buffer to the column matrix, and centrifuge at full speed to elute the DNA.

[0136] (10) Qubit 2.0 was used to detect the concentration of genomic DNA modified with bisulfite. The concentration of DNA after modification of each sample is shown in Table 4.

[0137] Table 4 DNA concentration after sample modification

[0138] sample Qubit (Salt to Concentration) sample-1 28ng / ul sample-2 34.8ng / ul sample-3 23.5ng / ul sample-4 29.3ng / ul sample-5 29.5ng / ul sample-6 27.7ng / ul

[0139] Example 3: Application of the primer and probe combination for detecting DNA methylation of OTX1 gene and PENK gene of the present invention in detecting bladder cancer

[0140] 1. Optimization and screening of the reaction system and reaction conditions for methylation fluorescence detection technology (Methylight)

[0141] Methylation fluorescence detection technology (MethyLight) is a quantitative fluorescence real-time PCR method, which is a detection technology that combines methyl-specific primers with methyl-specific fluorescence. This technology has a short detection cycle, high sensitivity, high repeatability, and a small sample size. It can quantitatively detect methylation with very low frequencies and can detect methylated alleles from more than 10,000 times the number of unmethylated alleles. It is a practical technology for early cancer screening.

[0142] In order to simplify the methylation detection method while ensuring high sensitivity and make it more suitable for clinical promotion and application, the present invention further verifies and optimizes the test experiment based on the Methylight technology by analyzing the previous sequencing results and public databases.

[0143] Using TaKaRa EpiTaq TM HS (for bisulfite-treated DNA) (Cat. No.: R110Q) was used for PCR reaction. For specific operations, please refer to the instructions of the kit.

[0144] The PCR reaction system used in the present invention is shown in Table 5.

[0145] Table 5 PCR reaction system of Methylight of the present invention

[0146] Reagents 20ul system Final concentration <![CDATA[ TaKaRa EpiTag HS(5U / ul )]]> 0.2ul <![CDATA[1.25U / 50 ul ]]> <![CDATA[10x EpiTaq PCR Buffer(Mg2+free)]]> 2ul 1X <![CDATA[25 mM MgCl2]]> 2ul <![CDATA[2.5 mM ]]> <![CDATA[ dNT P Mixture (2.5 each mM )]]> 2.4ul <![CDATA[0.3 mM ]]> Primer 1(10uM) 0.8ul <![CDATA[0.4 uM ]]> Primer 2(10uM) 0.8ul <![CDATA[0.4 uM ]]> Primer-probe 0.4ul <![CDATA[0.2 uM ]]> DNA template 2ul >50ng Sterilized water 9.4ul

[0147] In order to optimize and screen the PCR reaction conditions, the present invention tested the amplification of primers and probes under two different reaction conditions (two-step method and three-step method) in the same reaction system.

[0148] The PCR reaction conditions for the two-step method and the three-step method are shown in Table 6 and Table 7, respectively.

[0149] Table 6 PCR reaction conditions of the two-step method of the present invention

[0150]

[0151] Table 7 PCR reaction conditions of the three-step method of the present invention

[0152]

[0153] Under the same experimental conditions, the target genes OTX1 and PENK can be amplified in both the two-step method and the three-step method. The results of fluorescence PCR amplification showed that under the same reaction conditions, the three-step method had a smaller CT value in both the OTX1 gene and the PENK gene, indicating that under the same conditions, the three-step method reached the threshold cycle number earlier than the two-step method and had better amplification efficiency. The test results are shown in the attached Figure 4 As shown. Although the two-step method has more advantages in terms of time, the target gene has better amplification efficiency in the three-step method. In order to ensure the accuracy and reliability of the amplification results, the three-step method is a better detection condition.

[0154] 2. How to read the Ct value in the three-step test

[0155] In the three-step method, combined with the contents of Table 8, the Ct value is read using the following method:

[0156] Table 8 Average cycle number (CT) of OTX1 gene and PENK gene methylation cell lines and normal subjects

[0157]

[0158]

[0159] After PCR is completed, the baselines of βactin and the target area are adjusted respectively. The fluorescence value of the minimum Ct value of the sample in a PCR amplification 1-2 cycles before is set as the baseline value. The threshold is set at the inflection point of the S-shaped amplification curve to obtain the Ct value of each gene in the sample.

[0160] There is no amplification in the negative control, and the Ct value of the sample internal reference gene should be ≤35, indicating that the experimental results are valid and can be interpreted.

[0161] In the experiment, the positive reference value was determined based on the methylated cell line. When the Ct value of the target region in at least two of the three replicate wells was ≤40, the target region was determined to be methylation-positive in the sample; otherwise, the target region was determined to be methylation-negative in the sample.

[0162] 3. Application of the primer and probe combination of the present invention in detecting bladder cancer

[0163] Using the primers and probes designed in Example 1, the normal human urine genomic DNA was extracted according to the method of Example 2, and the extracted urine DNA and cell line DNA (positive reference) were modified with bisulfite. The samples were divided into a normal group (Normal) and a methylation group (Methylation), and the PCR reaction system of Example 3 was used to perform MethyLight detection according to the reaction conditions of the three-step method. Finally, the methylation was determined according to the standard in step 2 of Example 3.

[0164] The experimental results show that the OTX1 gene and the PENK gene can be used to distinguish between cell line samples and healthy samples. According to the determination method given in Example 3, all cell line test results are "positive" and all normal human test results are "negative".

[0165] The present application has been described above in conjunction with preferred embodiments, but these embodiments are only exemplary and serve only as an illustration. On this basis, various replacements and improvements may be made to the present application, all of which fall within the scope of protection of the present application.

Claims

1. A primer and probe combination, comprising the primer and probe combination described in groups 1-5, preferably consisting of the primer and probe combination described in groups 1-5, wherein group 1 consists of primers shown in SEQ ID NO.4-5 and probe shown in SEQ ID NO.6, group 2 consists of primers shown in SEQ ID NO.7-8 and probe shown in SEQ ID NO.9, and group 1 and group 2 are used to detect DNA methylation of OTX1 gene; group 3 consists of primers shown in SEQ ID NO.10-11 and probe shown in SEQ ID NO.12, group 4 consists of primers shown in SEQ ID NO.13-14 and probe shown in SEQ ID NO.15, and group 3 and group 4 are used to detect DNA methylation of PENK gene; group 5 consists of primers shown in SEQ ID NO.16-17 and probe shown in SEQ ID NO.18, and is used to detect DNA methylation of internal reference gene ACTB gene.

2. Use of the primer and probe combination according to claim 1 in preparing a kit.

3. A kit comprising the primer and probe combination according to claim 1.

4. The kit according to claim 3, further comprising a reagent for performing methylation fluorescence detection (Methylight) on the sample.

5. A PCR amplification method for methylation fluorescence detection (Methylight) of a sample, which is a three-step method, comprising the following steps: (1) 95°C, pre-denaturation, 5 minutes, (2) 95°C, denaturation, 15 seconds, (3) 60°C, annealing, 1 min; Step (2) and step (3) are repeated for 40 cycles. The primer and probe combination of claim 1 or the kit of claim 3 or 4 is used.

6. Use of the primer and probe combination of claim 1, the kit of claim 3 or 4, or the PCR amplification method of claim 5 in detecting DNA methylation of OTX1 gene and PENK gene.

7. A method for detecting DNA methylation of OTX1 gene and PENK gene, comprising the following steps: The primer and probe combination of claim 1, the kit of claim 3 or 4, or the PCR amplification method of claim 5 is used to detect the DNA methylation levels of the OTX1 gene and the PENK gene.

8. The method according to claim 7, comprising the steps of: (1) synthesizing the primer and probe combination described in claim 1, or using the primer and probe combination contained in the kit described in claim 3 or 4; (2) extracting sample DNA and performing bisulfite modification; (3) The PCR amplification method described in claim 5 is used to detect the DNA methylation levels of the OTX1 gene and the PENK gene. 9 . The method according to claim 7 or 8 , wherein the DNA methylation levels of the OTX1 gene and the PENK gene are detected by methylation fluorescence detection (Methylight) technology.

10. The method according to any one of claims 7 to 9, wherein the sample DNA is urine DNA from a normal person or a bladder cancer patient.

Images